Brazilian Journal of Microbiology (2008) 39:61-63

ISSN 1517-8382

ANTIFUNGAL ACTIVITY OF THE ESSENTIAL OIL FROM CALENDULA OFFICINALIS

L. (ASTERACEAE) GROWING IN BRAZIL

Zilda Cristiane Gazim1; Claudia Moraes Rezende2; Sandra Regina Fraga2; Terezinha Inez Estivaleti Svidzinski3;
Digenes Aparicio Garcia Cortez3*

1
Departamento de Farmcia, Universidade Paranaense, Umuarama, PR, Brasil; 2Instituto de Qumica, Universidade Federal do
Rio de Janeiro, Centro de Tecnologia, Rio de Janeiro, RJ, Brasil; 3Departamento de Farmcia e Farmacologia, Universidade
Estadual de Maring, Maring, PR, Brasil.

Submitted: April 23, 2007; Returned to authors for corrections: July 09, 2007; Approved: November 15, 2007.

SHORT COMMUNICATION

ABSTRACT

This study tested in vitro activity of the essential oil from flowers of Calendula officinalis using disk-
diffusion techniques. The antifungal assay results showed for the first time that the essential oil has good
potential antifungal activity: it was effective against all 23 clinical fungi strains tested.

Key words: Calendula officinalis; essential oil; antifungal activity

Calendula officinalis L. (Asteraceae) is an annual herb Plant material

with yellow to orange flowers, native to the Mediterranean
region. It is also known as pot marigold, a name historically
associated with its use in soups and stews to combat
illnesses (1) and has a long history of safe use as a medicine
in the treatment of inflammation and skin wounds (2). The
plant contains esquiterpenes glycosides, saponins,
xanthophylls, triol triterpenes, flavonoids and volatiles.
Chalchat and Cols (3) studied the essential oil of C. officinalis
flowers cultivated in the Massif Central, France, and obtained
sesquiterpene alcohol and mainly -cadinol by using steam
distillation. Radulescu and Cols (4) studied flowers from
Romania by headspace and steam distillation, and found -
cadinene plus 1,3,5-cadinatriene and -muurolol as the major
compounds, respectively. Because of the economic value of
C. officinalis as an herbal medicine and its wide use in
cosmetics, perfumes, pharmaceutical preparations and food,
we decided to study the acclimatization of C. officinalis in
southeast Brazil. The aim of the present work was to study in
vitro antifungal activity of the essential oil from C. officinalis
flowers, as determined by agar disk diffusion, on 23 clinical
fungal strains.
The Calendula officinalis flowers were collected from an
experimental plot in the Medicinal Botanical Garden of the
Universidade Paranaense in Umuarama, state of Paran,
southeastern Brazil (S23 46.225 and W 53 16.730, altitude 391
m). The flowers were dried at 25C in a lighted room for 20 days.
A voucher specimen, HEUP 1311, was deposited in the
Educational Herbarium of the Universidade Paranaense (HEUP).
The flowers were collected on 30 April 2004 (onset of winter).
Steam distillation
The essential oil was obtained in a Clevenger apparatus by
steam distillation. After 3 hours of steam distillation, l50 g of
dried sample was extracted with 500 ml of water. The water
collected was re-extracted with 3 x 50 ml hexane. After drying in
anhydrous Na2SO4, hexane was concentrated in a vacuum rotator
evaporator apparatus to 47 mg to yield 0.1% w/w by weight of
dry material (5).
Microorganisms used and growth conditions
The antimicrobial activity of the essential oil from Calendula
officinalis flowers was evaluated using a panel which included

*Corresponding Author. Mailing address: Departament of Pharmacy and Pharmacology, Universidade Estadual de Maring, Av. Colombo, 5790,
87020-900, Maring, PR, Brazil. E-mail: dagcortez@uem.br

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Gazim, Z.C. et al.

laboratory control strains from the American Type Culture essential oils rich in cadinene isomers are widely reported to
Collection (Rockville, MD, USA): fungal microorganisms: possess high levels of anticandidal activity (10). Our data
Candida albicans (ATCC 64548), Candida dubliniensis (ATCC indicate that the oil of C. officinalis flowers exhibited antifungal
777), Candida parapsilosis (ATCC 22019), Candida glabrata activity against all investigated yeasts, including the clinical
(ATCC 90030) and Candida krusei (ATCC 6258); and the pathogens obtained from reference stocks as well as those
following yeasts clinically isolated from humans: Candida recently isolated. This oil provided more interesting results
albicans, Candida dubliniensis, Candida parapsilosis, than Nystatin, a fungicide used for topical treatment of
Candida glabrata, Candida tropicalis, Candida mucocutaneous candidiasis.
guilliermondii, Candida krusei and Rhodotorulla sp. The As seen in Table 1, discs impregnated with 15 g of C.
yeasts were cultured at 25C in Sabouraud dextrose agar. officinalis flower oil produced inhibition zones ranging from 11
to 30 mm of the diameter. The widest (28-30 mm) were obtained
Disc diffusion method against Candida parapsilosis (isolates 11 and 12), Candida
In vitro antifungal activity of the C. officinalis essential oil glabrata (isolate 15) and Rhodotorulla sp. (isolate 23). The oil
was determined by the agar disk diffusion method according to also showed high activity, with inhibition zones of 20-27 mm,
Rubio et al. 2003 (6). Briefly, a suspension of each tested against Candida albicans (Isolates 3 and 7), Candida
microorganism (2.0 ml of 105 cells per ml) was
carefully mixed in a tube with 18 ml of Mueller
Hinton Agar (MHA), and then poured on Petri Table 1. Antifungal activities of the essential oil of flowers of Calendula
plates. Sterile filter-paper discs (Whatman No. officinalis.
1, 6.0 mm in diameter) were impregnated with
15 l of the oil and placed on the inoculated Mean zone of inhibition
plates. Control disks containing 15 l of the a (mm)
Isolate Microorganisms Origin*
physiological saline and Nystatin (100 U.I. or Calendula Nystatin
20 g/disc, Cecon, So Paulo, Brazil) were oil15 l/disc 20 g/disc
used. These plates were allowed to dry at room
temperature for 2 h, and were incubated at 25C 1 C. albicans ATCC 64548 16 12
for 48 h. The diameters of the inhibition zones 2 C. albicans orotracheal tube 11 13
were measured in millimeters and their means 3 C. albicans OC HIV 26 11
were calculated. All the tests were performed 4 C. albicans VVC 18 12
in duplicate (7). Twenty-three yeast strains 5 C. albicans VVC 15 12
were tested, as listed in Table 1. 6 C. albicans VVC 15 12
The main constituents of the essential oil 7 C. albicans Urine 27 11
were the following: sesquiterpene 8 C dubliniensis ATCC 777 24 11
hydrocarbons (68.0%) and sesquiterpenols 9 C. parapsilosis ATCC 22019 20 12
(27.0%). -cadinene (22.53%), -cadinol 10 C. parapsilosis Onychomycosis 14 13
(20.40%) and epi--muurolol (12.87%). The 11 C. parapsilosis Paronychia 30 11
analyses were performed by GC and GC-MS 12 C. parapsilosis Blood 30 11
as described by Gazim et al. (8). 13 C. glabrata ATCC 90030 15 12
Many antifungal agents are available for 14 C. glabrata Hands colonization 23 11
the treatment of candidal infections, and 15 C. glabrata Hands colonization 28 11
these are available in several pharmaceutical 16 C. tropicalis Urine 11 13
forms for either topical or systemic use. The 17 C. tropicalis Granulomatous lesion 15 12
major agents belong either to the polyenes, 18 C. tropicalis Urine 21 12
such as amphotericin B and nystatin; or to 19 C. tropicalis Urine 22 11
the azoles, such as itraconazole and 20 C. guilliermondii Hands colonization 25 11
fluconazole. However, because of the need 21 C. guilliermondii Hands colonization 24 11
for extended treatment, the high cost, toxicity 22 C. krusei ATCC 6258 15 12
and limited action of the classic drugs, new 23 Rhodotorulla sp Hands colonization 30 11
and effective products are desirable to treat * Except to ATCC microorganisms all of others are human clinical isolates OC HIV: oral
these fungal infections. The antifungal effect candidiasis; VVC: vulvovaginal candidiasis. Mean of inhibition zone by oil of flowers of
of essential oils (EO) of many aromatic plants Calendula officinalis: Good activity (11 -18 mm); high activity (20-27 mm); highest
has been described in several studies (9). The activity (28-30 mm).

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dubliniensis ATCC 777, Candida parapsilosis ATCC 22019,
Candida tropicalis (Isolates 18 and 19), Candida guilliermondii
(Isolates 20 and 21) and Candida glabrata (Isolate 14). For ten
isolates: Candida albicans ATCC 64548 and isolates 2, 4, 5 and
6, Candida parapsilosis (Isolate 10), Candida glabrata ATCC
90030, Candida tropicalis (Isolates 16 and 17) and Candida
krusei ATCC 6258, this oil also showed good antifungal activity
(11-18 mm).
We note that according to the manufacturer of the Nystatin
disks, all 23 samples of yeasts tested were sensitive to Nystatin
(inhibition diameter above 10 mm). However, the oil of C.
officinalis flowers showed greater variability among the different
isolates than did Nystatin, which ranged between 11 and 13
mm. It is possible that the wider range in the sensitivity profile
shown by the oil of C. officinalis flowers may be advantageous,
because these are widely available and demonstrate a wide action
spectrum against pathogenic fungi. Moreover, the human
therapeutic response to medicine is not uniform, as suggested
by the in vitro assays with Nystatin.
ACKNOWLEDGEMENTS
The authors thank the Coordenao de Aperfeioamento
de Pessoal de Nvel Superior (CAPES) and the Conselho
Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq)
for financial support.
RESUMO
Atividade antifngica do leo essencial da Calendula
officinalis cultivada no Brasil
Neste estudo foi avaliada a atividade antifngica do leo
essencial, obtido das flores de Calendula officinalis utilizando-
se tcnica de difuso em discos. Os resultados dos ensaios
antifngicos mostraram pela primeira vez, que o leo essencial
Antifungal activity of C. officinalis
tem um grande potencial antifngico, pois foi efetivo contra
todas as 23 amostras clnicas de fungos testados.
Palavras-chave: Calendula officinalis; leo essencial; ativida-
de antifngica
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